It is frequently necessary to confirm the presence of drug substances, their metabolites, etc., in serum or plasma and/or to measure the concentrations of these compounds. In other cases, it is necessary to separate bio-polymers from smaller substances as a step in purifying substances from biological or from biomass mixtures. Such analyses are carried out using liquid chromatographic systems as illustrated in FIG. 1 of the drawings. The invention is compatible with high performance liquid chromatographic systems but is not limited to them.
Most of the published data and methods in this area of research relate to the LC analysis of drugs, metabolites, etc., in serum or plasma. The ways the mixtures are sampled can be classified as indirect and direct sampling. Indirect sampling involves treatment of the sample to remove the proteins, e.g. by precipitation, followed by extraction of the compound(s) of interest into a protein-free solvent system. Although this method involves multi-step preparation before the sample can be analyzed by a particular LC method, it still attracts much of the practical attention. Direct sampling, or direct injection of the untreated sample on an LC analytical column, causes clogging of the column, resulting in increasing pressure drop, peak broadening, variation of retention times, etc., unless special precautions are taken. After each sample injection, or after a few injections, the column must be thoroughly washed to remove precipitated proteins, particularly when larger serum samples (.gtoreq.10 .mu.l) are needed to detect the analytes of interest at their therapeutic or biological levels.
A partial solution to the above problems was found in a combination of an analytical column and a precolumn and two delivery pumps in a column switching system. Usually, the serum sample is loaded onto a short precolumn under mobile phase conditions in which only the drug(s) elute onto the analytical column. When all the components of interest elute from the precolumn to the analytical column, a valve is switched so that one pump continues to deliver mobile phase for elution of the compounds of interest from the analytical column for separation, while the second pump delivers a washing solution to the precolumn for removal of the proteins. To avoid clogging, the precolumn is filled with relatively large particles, usually 20-40 .mu.m, and is replaced frequently to avoid deterioration of the analytical column (W. Rothe, et al., J. Chromatog. 222 (1981) 13). Usually, both columns are filled with reversed phase packing, e.g. C.sub.8 or C.sub.18 bonded to a silica support.
To avoid protein accumulation in the precolumn and to speed up the washing step, a less hydrophobic packing has been used, butyl modified methacrylate, as manufactured by TosoH, Japan, and sold under the tradename TOYOPEARL.TM. BT-650M. In the loading cycle, 10-50% saturated ammonium sulfate (NH.sub.4).sub.2 SO.sub.4 aqueous mobile phase is used. Under such conditions, serum proteins are retained on the precolumn and the drugs elute to load the reversed phase analytical column. Then, by column switching, the analytical column is separately programmed, while the precolumn is cleaned of the retained proteins, using a buffer solution of lower ionic strength (G. Tamai, et al., Chromatographia 21 (1986) 519).
In another study, a polystyrene divinylbenzene resin, manufactured by Rohm and Haas, USA, and sold under the tradename Amberlite.RTM. XAD-2, was used as the packing in the precolumn to retain methaqualone (MTQ), while eluting the plasma proteins. After all the proteins are washed away (with a pH 9.3 buffer solution), the mobile phase is adjusted to elute MTQ (R. A. Hux, et al., Anal. Chem. 34 (1982) 113).
Another example of a two-modal HPLC system combines size exclusion chromatography (SEC) and reversed phase chromatography (RPLC) using two columns in a column switching system. Following exclusion of the biopolymers from the SEC column, the later eluting band of smaller molecular size compounds was backflushed to the RPLC analytical column (S. F. Chang, et al., J. Pharm. Sci. 72 (1983) 236).
All the above examples employ column switching which requires an elaborate chromatographic system, including a second solvent delivery system, a second column and a switching system. Moreover, the operation of the switching system itself requires labor or investment in additional control equipment.
A completely different approach was undertaken by Pinkerton, et al. (U.S. Pat. No. 4,544,485). They redesigned the packing of the analytical column in such a way that the proteins elute in the excluded volume (void volume) and the analytes are retained and separated on the same analytical column. This was accomplished by chemically modifying a hydrophilic diol phase with a hydrophobic oligopeptide, e.g. glycyl-(L-phenylalanine)n, where n=1,2, or 3. It is crucial to their invention that the diol phase is bonded to a porous silica gel having a pore diameter smaller than 80 angstroms. Following this modification, the phenylalanine moiety is enzymatically cleaved from the diol ligand with a protease. The cleavage is restricted to surface areas that are accessible to the protease, resulting in a support for which the diol ligands are only present on the external surface, while L-phenylalanine modified ligands are present in the internal surface, i.e., the pores of the packing material. The ligands that are not accessible to the enzyme are similarly not accessible to the serum proteins. Thus, these proteins are excluded from entering the pores and elute in the void volume, while the smaller molecules (e.g., drugs) can interact with the hydrophobic phenylalanine ligands (U.S. Pat. No. 4,544,485). This support, named internal surface reverse phase liquid chromatographic packing (IS-RP), can be used to analyze many serum sample without the damaging accumulation of proteinaceous precipitate seen on regular RPLC columns.
Conceptually, the study of Yoshida, et al., (Chromatographia 19 (1985) 466) is similar to that of Pinkerton. They adsorbed denatured plasma proteins on C.sub.18 silica supports having small pore diameter. These supports no longer retained plasma proteins, but still showed reversed phase characteristics for smaller analytes. The phenomenon is depicted as similar to that of Pinkerton's model, or as having the proteinaceous precipitation limited to the externally exposed surface, thereby making the external surface hydrophilic, while keeping the non-exposed internal C.sub.18 surface free of such precipitation and accessible for (hydrophobic) interaction with small compounds.
Thus an object of this invention is to provide a novel packing material for liquid chromatography which will allow the direct injection of biological fluids into the column.
Another object of this invention is to provide a packing material for chromatographic columns which has a hydrophilic exterior layer and a hydrophobic underlayer.
Still another object of this invention is to provide a chromatographic column which will shield and exclude large biopolymers but permit the partitioning of and hydrophobic interaction with small analytes.
Yet another object of this invention is to provide a novel shielded hydrophobic phase packing for chromatography adapted to bond to porous and non-porous silica supports.
Another object of this invention is to provide a chromatographic phase having a covalently bonded micellar surface.
Another object of this invention is to provide a packing material for chromatographic columns which has a hydrophilic exterior layer and an anionic underlayer.
Another object of this invention is to provide a packing material for chromatographic columns which has a hydrophilic exterior layer and a cationic underlayer.
Another object of this invention is to provide a packing material for chromatographic columns which has a hydrophilic exterior layer and a chelating underlayer.
These and other objects of this invention may be seen by reference to the present specifications, claims, and drawings.